Glaucoma Animal Models beyond Chronic IOP Increase.

Glaucoma is a complex and multifactorial disease defined as the loss of retinal ganglion cells (RGCs) and their axons. Besides an elevated intraocular pressure (IOP), other mechanisms play a pivotal role in glaucoma onset and progression. For example, it is known that excitotoxicity, immunological alterations, ischemia, and oxidative stress contribute to the neurodegeneration in glaucoma disease. To study these effects and to discover novel therapeutic approaches, appropriate animal models are needed. In this review, we focus on various glaucoma animal models beyond an elevated IOP. We introduce genetically modified mice, e.g., the optineurin E50K knock-in or the glutamate aspartate transporter (GLAST)-deficient mouse. Excitotoxicity can be mimicked by injecting the glutamate analogue N-methyl-D-aspartate intravitreally, which leads to rapid RGC degeneration. To explore the contribution of the immune system, the experimental autoimmune glaucoma model can serve as a useful tool. Here, immunization with antigens led to glaucoma-like damage. The ischemic mechanism can be mimicked by inducing a high IOP for a certain amount of time in rodents, followed by reperfusion. Thereby, damage to the retina and the optic nerve occurs rapidly after ischemia/reperfusion. Lastly, we discuss the importance of optic nerve crush models as model systems for normal-tension glaucoma. In summary, various glaucoma models beyond IOP increase can be utilized.

Etiology of Idiopathic Macular Holes in the Light of Estrogen Hormone.

The aim of this review was to identify a new potential explanation for the development of macular holes in relation to the female sex and to explain the possible underlying pathways. This approach was based on the evaluation of anatomical, physiological, and morphological analyses currently available in the literature. The findings showed that estrogen exerts a protective effect on the neuroretina and may influence Müller and cone cells. Both cell types are responsible for the building of the fovea structure. However, this protection may be lost due to the sudden decrease in estrogen levels during menopause. In conclusion, the fovea cones, through its sensitivity to estrogen and high energy consumption, may be very vulnerable to damage caused by a sudden changes in the concentration of estrogen in menopausal females. Such changes may result in cone degeneration, and thus a destroyed structure of the fovea, and may lead to the development of a hole in the fovea, as in the case of macular holes. This review revealed that under the decreasing influence of estrogen may cones play a key role with regard to the etiology of the development of macular holes. This aspect may be of strategic importance in prophylactic therapy for the prevention of the development of macular holes in premenopausal females or after ocular trauma.

Islet Co-Expression of CD133 and ABCB5 in Human Retinoblastoma Specimens.

BACKGROUND: The role of CD133 und ABCB5 is discussed in treatment resistance in several types of cancer. The objective of this study was to evaluate whether CD133+/ABCB5+ colocalization differs in untreated, in beam radiation treated, and in chemotherapy treated retinoblastoma specimens. Additionally, CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 gene expression was analyzed in WERI-RB1 (WERI RB1) and etoposide-resistant WERI RB1 subclones (WERI ETOR). METHODS: Active human untreated retinoblastoma specimens (n = 12), active human retinoblastoma specimens pretreated with beam radiation before enucleation (n = 8), and active human retinoblastoma specimens pretreated with chemotherapy before enucleation (n = 7) were investigated for localization and expression of CD133 and ABCB5 by immunohistochemistry. Only specimens with IIRC D, but not E, were included in this study. Furthermore, WERI RB1 and WERI ETOR cell lines were analyzed for CD133, ABCB5, sphingosine kinase 1, and sphingosine kinase 2 by the real-time polymerase chain reaction (RT-PCR). RESULTS: Immunohistochemical analysis revealed the same amount of CD133+/ABCB5+ colocalization islets in untreated and treated human retinoblastoma specimens. Quantitative RT-PCR analysis showed a statistically significant upregulation of CD133 in WERI ETOR (p = 0.002). No ABCB5 expression was detected in WERI RB1 and WERI ETOR. On the other hand, SPHK1 (p = 0.0027) and SPHK2 (p = 0.017) showed significant downregulation in WERI ETOR compared to WERI RB1. CONCLUSIONS: CD133+/ABCB5+ co-localization islets were noted in untreated and treated human retinoblastoma specimens. Therefore, we assume that CD133+/ABCB5+ islets might play a role in retinoblastoma genesis, but not in retinoblastoma treatment resistance.

Smad7 in intestinal CD4+ T cells determines autoimmunity in a spontaneous model of multiple sclerosis.

Environmental triggers acting at the intestinal barrier are thought to contribute to the initiation of autoimmune disorders. The transforming growth factor beta inhibitor Smad7 determines the phenotype of CD4+ T cells. We hypothesized that Smad7 in intestinal CD4+ T cells controls initiation of opticospinal encephalomyelitis (OSE), a murine model of multiple sclerosis (MS), depending on the presence of gut microbiota. Smad7 was overexpressed or deleted in OSE CD4+ T cells to determine the effect on clinical progression, T cell differentiation, and T cell migration from the intestine to the central nervous system (CNS). Smad7 overexpression worsened the clinical course of OSE and increased CNS inflammation and demyelination. It favored expansion of intestinal CD4+ T cells toward an inflammatory phenotype and migration of intestinal CD4+ T cells to the CNS. Intestinal biopsies from MS patients revealed decreased transforming growth factor beta signaling with a shift toward inflammatory T cell subtypes. Smad7 in intestinal T cells might represent a valuable therapeutic target for MS to achieve immunologic tolerance in the intestine and suppress CNS inflammation.

Coculture of ARPE-19 Cells and Porcine Neural Retina as an Ex Vivo Retinal Model.

Neural retinal organ cultures are used to investigate ocular pathomechanisms. However, these cultures lack the essential retinal pigment epithelium (RPE) cells, which are part of the actual in vivo retina. To simulate a more realistic ex vivo model, porcine neural retina explants were cocultured with ARPE-19 cells (ARPE-19 group), which are derived from human RPE. To identify whether the entire cells or just the cell factors are necessary, in a second experimental group, porcine neural retina explants were cultured with medium derived from ARPE-19 cells (medium group). Individually cultured neural retina explants served as controls (control group). After 8 days, all neural retinas were analysed to evaluate retinal thickness, photoreceptors, microglia, complement factors and synapses (n = 6-8 per group). The neural retina thickness in the ARPE-19 group was significantly better preserved than in the control group (p = 0.031). Also, the number of L-cones was higher in the ARPE-19 group, as compared to the control group (p < 0.001). Furthermore, the ARPE-19 group displayed an increased presynaptic glutamate uptake (determined via vGluT1 labelling) and enhanced post-synaptic density (determined via PSD-95 labelling). Combined Iba1 and iNOS detection revealed only minor effects of ARPE-19 cells on microglial activity, with a slight downregulation of total microglia activity apparent in the medium group. Likewise, only minor beneficial effects on photoreceptors and synaptic structure were found in the medium group. This novel system offers the opportunity to investigate interactions between the neural retina and RPE cells, and suggests that the inclusion of a RPE feeder layer has beneficial effects on the ex vivo maintenance of neural retina. By modifying the culture conditions, this coculture model allows a better understanding of photoreceptor death and photoreceptor-RPE cell interactions in retinal diseases.

Towards an Understanding of Retinal Diseases and Novel Treatment.

The Special Issue, "Towards an Understanding of Retinal Diseases and Novel Treatment", provides comprehensive information on retinal diseases such as glaucoma, age-related macular degeneration (AMD), diabetic retinopathy, retinitis pigmentosa (RP), and others [...].

Changes of Subjective Symptoms and Tear Film Biomarkers following Femto-LASIK.

Femtosecond laser-assisted in situ keratomileusis (Femto-LASIK) represents a common treatment modality in refractive surgery and shows excellent results in terms of safety, efficacy, predictability, and long-term stability. However, patients may be affected by dry eye symptoms. The aim of this study was to identify a potential association between subjective dry eye symptoms, objective dry eye markers, and possible changes in the tear film, which could be a target for future therapy development. Therefore, clinical (dry eye) examinations (OSDI, Schirmer test, lissamine green and fluorescein staining, BUT, visual acuity) were carried out before LASIK as well as 5 and 90 days post-OP. The dry eye marker MMP-9, cytokines (IL-1ß, IL-8), and pain markers (NGF, CGRP) were quantified in tear samples with immunoassays. In addition, correlation analyses were performed. Clinical examinations revealed an upregulated OSDI score 5 days post-OP and an increased lissamine green staining score 90 days post-OP. Downregulated CGRP levels were noted 5 days post-OP, while other protein markers were not significantly altered after Femto-LASIK. Hence, Femto-LASIK surgery induced subjective symptoms like that of dry eye which could objectively rather be classified as Femto-LASIK-related discomfort. In the future, this could possibly be better detected and treated using pain markers such as CGRP.

Protein Profiling of WERI-RB1 and Etoposide-Resistant WERI-ETOR Reveals New Insights into Topoisomerase Inhibitor Resistance in Retinoblastoma.

Chemotherapy resistance is one of the reasons for eye loss in patients with retinoblastoma (RB). RB chemotherapy resistance has been studied in different cell culture models, such as WERI-RB1. In addition, chemotherapy-resistant RB subclones, such as the etoposide-resistant WERI-ETOR cell line have been established to improve the understanding of chemotherapy resistance in RB. The objective of this study was to characterize cell line models of an etoposide-sensitive WERI-RB1 and its etoposide-resistant subclone, WERI-ETOR, by proteomic analysis. Subsequently, quantitative proteomics data served for correlation analysis with known drug perturbation profiles. Methodically, WERI-RB1 and WERI-ETOR were cultured, and prepared for quantitative mass spectrometry (MS). This was carried out in a data-independent acquisition (DIA) mode. The raw SWATH (sequential window acquisition of all theoretical mass spectra) files were processed using neural networks in a library-free mode along with machine-learning algorithms. Pathway-enrichment analysis was performed using the REACTOME-pathway resource, and correlated to the molecular signature database (MSigDB) hallmark gene set collections for functional annotation. Furthermore, a drug-connectivity analysis using the L1000 database was carried out to associate the mechanism of action (MOA) for different anticancer reagents to WERI-RB1/WERI-ETOR signatures. A total of 4756 proteins were identified across all samples, showing a distinct clustering between the groups. Of these proteins, 64 were significantly altered (q < 0.05 & log2FC |>2|, 22 higher in WERI-ETOR). Pathway analysis revealed the "retinoid metabolism and transport" pathway as an enriched metabolic pathway in WERI-ETOR cells, while the "sphingolipid de novo biosynthesis" pathway was identified in the WERI-RB1 cell line. In addition, this study revealed similar protein signatures of topoisomerase inhibitors in WERI-ETOR cells as well as ATPase inhibitors, acetylcholine receptor antagonists, and vascular endothelial growth factor receptor (VEGFR) inhibitors in the WERI-RB1 cell line. In this study, WERI-RB1 and WERI-ETOR were analyzed as a cell line model for chemotherapy resistance in RB using data-independent MS. Analysis of the global proteome identified activation of "sphingolipid de novo biosynthesis" in WERI-RB1, and revealed future potential treatment options for etoposide resistance in RB.

Specific Biomarkers in the Aqueous Humour of Glaucoma Patients. / Spezifische Biomarker im Kammerwasser von Glaukompatienten.

Glaucoma, a multifactorial neurodegenerative disease, is the second most common cause of blindness. Since early diagnosis facilitates timely treatment, it is therefore essential to identify appropriate markers. In the future, so-called biomarkers could be helpful in early detection and follow-up. In glaucoma, these parameters could be obtained in the aqueous humour. Altered antibodies, proteins, microRNA (miRNA) and trace element levels have already been identified. This review provides insight into possible changes in the aqueous humour of patients with primary open-angle glaucoma (POAG), normal tension glaucoma (NTG) or pseudoexfoliation glaucoma (PEXG). Studies on antibody changes in POAG patients identified an upregulation of immune system associated antibodies such as heat shock protein (HSP) 27. HSP27 was also upregulated in PEXG patients but decreased in NTG. In POAG and PEXG samples, the levels of certain proteins, including interleukins and endothelin-1, were elevated. The vasoconstrictor endothelin-1 may play a role in regulating intraocular pressure. By contrast, proteins playing a role in the response to oxidative stress were downregulated. In NTG patients, proteins responsible for the elimination of toxic by-products from the respiratory chain were downregulated. In addition, the aqueous humour of POAG and PEXG patients contained several miRNAs that have been linked to tissue development, neurological disease and cellular organisation. Other miRNAs regulated in glaucoma play a role in extracellular matrix remodelling and thus may affect drainage resistance in the trabecular meshwork. It is also interesting to note that the aqueous humour of glaucoma patients showed changes in the levels of trace elements such as zinc and selenium. The elevated zinc levels could be responsible for the imbalance of intraocular matrix metalloproteinases and thus for increased intraocular pressure. All these studies demonstrate the complex changes in aqueous humour in glaucoma. Some of these biomarkers may be useful in the future for early diagnosis of the disease.

Age-dependent favorable visual recovery despite significant retinal atrophy in pediatric MOGAD: how much retina do you really need to see well?

BACKGROUND: To investigate age-related severity, patterns of retinal structural damage, and functional visual recovery in pediatric and adult cohorts of myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) optic neuritis (ON). METHODS: All MOGAD patients from the 5 participating centers were included. Patients with initial manifestation <18 years were included in the pediatric (MOGADped) cohort and patients with ≥18 years in the adult (MOGADadult) cohort. For patients with MOGAD ON, examinations at least ≥6 months after ON onset were included in the analyses. Using spectral domain optical coherence tomography (SD-OCT), we acquired peripapillary retinal nerve fiber layer thickness (pRNFL) and volumes of combined ganglion cell and inner plexiform layer (GCIPL). High- and 2.5% low-contrast visual acuity (HCVA, LCVA) and visual-evoked potentials (VEP) were obtained. RESULTS: Twenty MOGADped (10.3±3.7 years, 30 MOGAD ON eyes) and 39 MOGADadult (34.9±11.6 years, 42 MOGAD ON eyes) patients were included. The average number of ON episodes per ON eye was similar in both groups (1.8±1.3 and 2.0±1.7). In both pediatric and adult MOGAD, ON led to pronounced neuroaxonal retinal atrophy (pRNFL: 63.1±18.7 and 64.3±22.9 µm; GCIPL: 0.42±0.09 and 0.44±0.13 mm3, respectively) and moderate delay of the VEP latencies (117.9±10.7 and 118.0±14.5 ms). In contrast, visual acuity was substantially better in children (HCVA: 51.4±9.3 vs. 35.0±20.6 raw letters, p=0.001; LCVA: 22.8±14.6 vs. 13.5±16.4, p=0.028). Complete visual recovery (HCVA-logMAR 0.0) occurred in 73.3% of MOGADped and 31% MOGADadults ON eyes, while 3.3% and 31% demonstrated moderate to severe (logMAR > 0.5) visual impairment. Independent of retinal atrophy, age at ON onset significantly correlated with visual outcome. CONCLUSION: Pediatric MOGAD ON showed better visual recovery than adult MOGAD ON despite profound and almost identical neuroaxonal retinal atrophy. Age-related cortical neuroplasticity may account for the substantial discrepancy between structural changes and functional outcomes.

Heat Shock Protein 27 Injection Leads to Caspase Activation in the Visual Pathway and Retinal T-Cell Response.

Heat shock protein 27 (HSP27) is one of the small molecular chaperones and is involved in many cell mechanisms. Besides the known protective and helpful functions of intracellular HSP27, very little is known about the mode of action of extracellular HSP27. In a previous study, we showed that intravitreal injection of HSP27 led to neuronal damage in the retina and optic nerve after 21 days. However, it was not clear which degenerative signaling pathways were induced by the injection. For this reason, the pathological mechanisms of intravitreal HSP27 injection after 14 days were investigated. Histological and RT-qPCR analyses revealed an increase in endogenous HSP27 in the retina and an activation of components of the intrinsic and extrinsic apoptosis pathway. In addition, an increase in nucleus factor-kappa-light-chain-enhancer of activated B cells (NFκB), as well as of microglia/macrophages and T-cells could be observed. In the optic nerve, however, only an increased apoptosis rate was detectable. Therefore, the activation of caspases and the induction of an incipient immune response seem to be the main triggers for retinal degeneration in this intravitreal HSP27 model.

Activation of Apoptosis in a ßB1-CTGF Transgenic Mouse Model.

To reveal the pathomechanisms of glaucoma, a common cause of blindness, suitable animal models are needed. As previously shown, retinal ganglion cell and optic nerve degeneration occur in ßB1-CTGF mice. Here, we aimed to determine possible apoptotic mechanisms and degeneration of different retinal cells. Hence, retinae were processed for immunohistology (n = 5-9/group) and quantitative real-time PCR analysis (n = 5-7/group) in 5- and 10-week-old ßB1-CTGF and wildtype controls. We noted significantly more cleaved caspase 3+ cells in ßB1-CTGF retinae at 5 (p = 0.005) and 10 weeks (p = 0.02), and a significant upregulation of Casp3 and Bax/Bcl2 mRNA levels (p < 0.05). Furthermore, more terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL+) cells were detected in transgenic mice at 5 (p = 0.03) and 10 weeks (p = 0.02). Neurofilament H staining (p = 0.01) as well as Nefh (p = 0.02) and Tubb3 (p = 0.009) mRNA levels were significantly decreased at 10 weeks. GABAergic synapse intensity was lower at 5 weeks, while no alterations were noted at 10 weeks. The glutamatergic synapse intensity was decreased at 5 (p = 0.007) and 10 weeks (p = 0.01). No changes were observed for bipolar cells, photoreceptors, and macroglia. We conclude that apoptotic processes and synapse loss precede neuronal death in this model. This slow progression rate makes the ßB1-CTGF mice a suitable model to study primary open-angle glaucoma.

Minocycline reduces inflammatory response and cell death in a S100B retina degeneration model.

BACKGROUND: Previous studies noted that intravitreal injection of S100B triggered a glaucoma-like degeneration of retina and optic nerve as well as microglia activation after 14 days. The precise role of microglia in our intravitreal S100B model is still unclear. Hence, microglia were inhibited through minocycline. The aim is to investigate whether microglia have a significant influence on the degeneration process or whether they are only a side effect in the model studied here. METHODS: Minocycline was applied daily in rats by intraperitoneal injection using two different concentrations (13.5 mg/kg body weight, 25 mg/kg body weight). One day after treatment start, S100B or PBS was intravitreally injected in one eye per rat. The naïve groups received no injections. This resulted in a total of five groups (naïve n = 14, PBS n = 14, S100B n = 13, 13.5 mg/kg mino n = 15, 25 mg/kg mino n = 15). At day 14, electroretinogram measurements were performed, followed by immunofluorescence and label-free quantitative proteomics analysis. The focus of these investigations was on the survival of RGCs as well as their axons, the response of the microglia, and the identification of further pathological modes of action of S100B. RESULTS: The best signal transmission was detected via ERG in the 13.5 mg/kg mino group. The inhibition of the microglia protected optic nerve neurofilaments and decreased the negative impact of S100B on RGCs. However, the minocycline treatment could not trigger complete protection of RGCs. Furthermore, in retina and optic nerve, the minocycline treatment reduced the number and activity of S100B-triggered microglia in a concentration-dependent manner. Proteomics analysis showed that S100B application led to numerous metabolic functions and cellular stress, mainly an increased inflammatory response, glycolysis, and mitochondrial dysfunction, which caused oxidative stress in the retina. Importantly, the protective capability of lower dose of minocycline was unraveled by suppressing the apoptotic, inflammatory, and the altered metabolic processes caused by S100B insult in the retina. CONCLUSION: Intravitreally injected S100B not only led to a pro-inflammatory microglial reaction, but also a mitochondrial and metabolic dysfunction. Also, these results suggest that an excessive microglial response may be a significant degenerative factor, but not the only trigger for increased cell death.

Preservation of optic nerve structure by complement inhibition in experimental glaucoma.

Glaucoma is characterized by a progressive damage of the retina and the optic nerve. Despite a huge research interest, the exact pathomechanisms are still unknown. In the experimental autoimmune glaucoma model, rats develop glaucoma-like damage of the retina and the optic nerve after immunization with an optic nerve antigen homogenate (ONA). An early activation of the complement system, even before optic nerve degeneration, was reported in this model. Here, we investigated the effects of a monoclonal antibody against complement factor C5 on optic nerves. Rats were immunized with ONA and compared to controls. In one eye of some ONA animals, the antibody against C5 was intravitreally injected (15 µmol: ONA + C5-I or 25 µmol: ONA + C5-II) before immunization and then every 2 weeks. After 6 weeks, optic nerves were processed for histology (n = 6/group). These analyses demonstrated that the intravitreal therapy reduced the depositions of the membrane attack complex compared to ONA animals (ONA + C5-I: p = 0.005; ONA + C5-II: p = 0.002). Cellular infiltration was significantly reduced in the ONA + C5-I group (p = 0.003), but not in ONA + C5-II tissues (p = 0.41). Furthermore, SMI-32 staining revealed that neurofilament was preserved in both treatment groups compared to ONA optic nerves (both p = 0.002). A decreased amount of microglia was found in treated animals in comparison to the ONA group (ONA + C5-I: p = 0.03; ONA + C5-II: p = 0.009). We observed, for the first time, that a complement system inhibition could prevent optic nerve damage in an autoimmune glaucoma model. Therefore, complement inhibition could serve as a new therapeutic tool for glaucoma.

Retinal ischemia triggers early microglia activation in the optic nerve followed by neurofilament degeneration.

Retinal ischemia leads to an early severe damage of the retina and thus plays an important role in eye diseases such as angle-closure glaucoma or retinal vascular occlusion. In retinal diseases, there is common sense about the affection of the optic nerve by ischemic injury. However, the exact dynamic processes of this optic nerve degeneration are mainly unclear. In this study, retinal ischemia was induced in one eye of Brown-Norway rats by raising the intraocular pressure 60 min to 140 mmHg followed by natural reperfusion. Optic nerves were analyzed at six different points in time: 2, 6, 12, and 24 h as well as 3 and 7 days after ischemic injury. Cell infiltration and moreover signs of tissue demyelination and dissolution were noticed in optic nerves 7 days after ischemia (hematoxylin & eosin: p < 0.001, luxol fast blue: p = 0.04). Although microglial activation was verified already from 12 h on after ischemia (p = 0.030), the beginning of a structural degeneration of the neurofilament was seen at 3 days (p = 0.02). Interestingly, proliferative microglia were present later on (7 days: p = 0.017). At this point, the number of total microglia was also increased in ischemic nerves (p = 0.003). Concluding, our data indicate that not only retinal tissue is affected by an ischemia, the optic nerve also demonstrates progressive damage. Interestingly, a microglia activation was noted days before structural damage became visible.

Protective effect of the extremolytes ectoine and hydroxyectoine in a porcine organ culture.

PURPOSE: Hypoxic damage to the retina is a relevant component of neurodegenerative pathologies such as glaucoma or retinal ischemia. In porcine retina organ cultures, hypoxic damage can be induced by applying cobalt chloride (CoCl2). The aim of our study was to investigate possible neuroprotective effects of the extremolytes ectoine and hydroxyectoine in this hypoxia-damaged retina model. METHODS: To simulate hypoxia, porcine retina organ cultures were damaged with 300 µM CoCl2 for 48 h starting on day 1 (n = 8-9/group). In order to investigate the possible neuroprotective effects of ectoine and hydroxyectoine, 0.5 mM of each extremolyte was added to the culture at the same time as the stressor and for the same duration. On day 8, the retina organ cultures were taken for (immuno)-histochemical examinations. Retinal ganglion cells (RGCs), macroglia, and apoptotic and hypoxic cells were detected with appropriate markers followed by cell counts and group comparisons. RESULTS: Treatment with ectoine resulted in RGC protection (p < 0.05) and reduced rate of apoptosis (p < 0.001) in hypoxia-treated retina organ cultures. However, the macroglia area and the amount of hypoxic, HIF-1α+ cells were unaffected by the ectoine treatment (p = 0.99). Treatment with hydroxyectoine also protected RGCs (p < 0.01) by inhibiting apoptosis (p < 0.001). In addition, the number of hypoxic, HIF-1α+ cells could be significantly reduced by treatment with hydroxyectoine (p < 0.05). The macroglia area on the other hand was unchanged after CoCl2 and treatment with hydroxyectoine. CONCLUSION: Both extremolytes had a protective effect on CoCl2-induced hypoxia in the porcine retina organ culture. Regarding the reduction of hypoxic stress, hydroxyectoine appears to be more effective. Thus, both extremolytes represent an interesting potential new therapeutic approach for patients with ocular diseases in which hypoxic processes play a significant role.

Oxidative stress-induced retinal damage is prevented by mild hypothermia in an ex vivo model of cultivated porcine retinas.

BACKGROUND: Hydrogen peroxide (H2 O2 ) can be used in vitro to simulate oxidative stress. In retinal organ cultures, H2 O2 induces strong neurodegeneration of the retina. It is known that oxidative stress plays a role in the development of several retinal diseases including glaucoma and ischemia. Thus, we investigated whether processes underlying oxidative stress can be prevented by hypothermia using an ex vivo organ culture model of porcine retinas. METHODS: Porcine retinal explants were cultivated for 5 and 8 days. Oxidative stress was induced via 300 µM H2 O2 on day 1 for 3 hours. Hypothermia treatment at 30°C was applied simultaneously with H2 O2 , for 3 hours. Retinal ganglion cells (RGCs), apoptosis, bipolar and cholinergic amacrine cells, microglia and macroglia were evaluated immunohistologically. Apoptosis rate was additionally analysed via western blot. RESULTS: Reduced apoptosis rates through hypothermia led to a preservation of RGCs (P < .001). Amacrine cells were rescued after hypothermia treatment (P = .17), whereas bipolar cells were only protected partly. Additionally, at 8 days, microglial response due to oxidative stress was completely counteracted via hypothermia (P < .001). CONCLUSIONS: H2 O2 induced strong degenerative processes in porcine retinas. The role of oxidative stress in the progression of retinal diseases makes this ex vivo organ culture model suitable to investigate new therapeutic approaches. In the present study, the damaging effect of H2 O2 to several retinal cell types was counteracted or strongly alleviated through hypothermia treatment. Especially RGCs, which are affected in glaucoma disease, were protected due to a reduced apoptosis rate through hypothermia.

[Work and Training of Young Doctors in Germany - a Comprehensive Analysis of Survey Results from Six Disciplines]. / Die Arbeits- und Weiterbildungssituation junger Ärztinnen und Ärzte in Deutschland ­ eine zusammenfassende Analyse von Befragungsergebnissen aus sechs Fachrichtungen.

OBJECTIVE: The working and training conditions of young physicians in Germany have changed over the last few years, as a result of far-reaching changes in the healthcare system. Therefore, Germany-wide surveys among young physicians of several disciplines were evaluated in a pooled analysis, in order to obtain a current interdisciplinary impression of conflicts in their daily work. MATERIAL AND METHODS: Data from web-based surveys from residents training in six disciplines were analyzed together retrospectively. One focus was a gratification crises model for the assessment of psychosocial workload. RESULTS: Data on 4041 participants were evaluated. In day-to-day work, young physicians were burdened with a high proportion of tasks that were not directly medical. Instruments of good subspecialty training, such as training contracts, curricula and regular feedback were associated with a lower psychosocial workload, which was generally significant among the participants. An economic influence on medical-professional decisions was subjectively clearly present among the participants. CONCLUSION: Many young doctors find the current work and training conditions in the medical work environment unsatisfactory. This might have consequences not only for the doctors themselves, but also for the patients they care for. A healthy work environment with health professionals in good health is therefore crucial and should be in everyone's interest.

Destructive Effect of Intravitreal Heat Shock Protein 27 Application on Retinal Ganglion Cells and Neurofilament.

Heat shock protein 27 (HSP27) is commonly involved in cellular stress. Increased levels of HSP27 as well as autoantibodies against this protein were previously detected in glaucoma patients. Moreover, systemic immunization with HSP27 induced glaucoma-like damage in rodents. Now, for the first time, the direct effects of an intravitreal HSP27 application were investigated. For this reason, HSP27 or phosphate buffered saline (PBS, controls) was applied intravitreally in rats (n = 12/group). The intraocular pressure (IOP) as well as the electroretinogram recordings were comparable in HSP27 and control eyes 21 days after the injection. However, significantly fewer retinal ganglion cells (RGCs) and amacrine cells were observed in the HSP27 group via immunohistochemistry and western blot analysis. The number of bipolar cells, on the other hand, was similar in both groups. Interestingly, a stronger neurofilament degeneration was observed in HSP27 optic nerves, while no differences were noted regarding the myelination state. In summary, intravitreal HSP27 injection led to an IOP-independent glaucoma-like damage. A degeneration of RGCs as well as their axons and amacrine cells was noted. This suggests that high levels of extracellular HSP27 could have a direct damaging effect on RGCs.

Synapse and Receptor Alterations in Two Different S100B-Induced Glaucoma-Like Models.

Glaucoma is identified by an irreversible retinal ganglion cell (RGC) loss and optic nerve damage. Over the past few years, the immune system gained importance in its genesis. In a glaucoma-like animal model with intraocular S100B injection, RGC death occurs at 14 days. In an experimental autoimmune glaucoma model with systemic S100B immunization, a loss of RGCs is accompanied by a decreased synaptic signal at 28 days. Here, we aimed to study synaptic alterations in these two models. In one group, rats received a systemic S100B immunization (n = 7/group), while in the other group, S100B was injected intraocularly (n = 6-7/group). Both groups were compared to appropriate controls and investigated after 14 days. While inhibitory post-synapses remained unchanged in both models, excitatory post-synapses degenerated in animals with intraocular S100B injection (p = 0.03). Excitatory pre-synapses tendentially increased in animals with systemic S100B immunization (p = 0.08) and significantly decreased in intraocular ones (p = 0.04). Significantly more n-methyl-d-aspartate (NMDA) receptors (both p ≤ 0.04) as well as gamma-aminobutyric acid (GABA) receptors (both p < 0.03) were observed in S100B animals in both models. We assume that an upregulation of these receptors causes the interacting synapse types to degenerate. Heightened levels of excitatory pre-synapses could be explained by remodeling followed by degeneration.